f u n g a l b i o l o g y 1 2 1 ( 2 0 1 7 ) 9 8 4e9 8 9

journa l homepage : www.e lsev ier . com/ loca te / funb io

Wood decay fungus Flavodon ambrosius(Basidiomycota: Polyporales) is widely farmed bytwo genera of ambrosia beetles

You LIa, Craig Christopher BATEMANb, James SKELTONa,Michelle Alice JUSINOc, Zachary John NOLENa, David Rabern SIMMONSd,Jiri HULCRa,b,*aSchool of Forest Resources and Conservation, University of Florida, Gainesville, FL, USAbDepartment of Entomology and Nematology, University of Florida, Gainesville, FL, USAcUnited States Forest Service, Northern Research Station, Center for Forest Mycology Research, Madison, WI, USAdDepartment of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI, USA

a r t i c l e i n f o

Article history:

Received 18 April 2017

Received in revised form

1 August 2017

Accepted 18 August 2017

Available online 26 August 2017

Corresponding Editor:

Alga Zuccaro

Keywords:

Ambrosia beetle

Ambrosiodmus

Ambrosiophilus

Nuclear 28S rDNA

Nuclear ITS rDNA

White rot

* Corresponding author. School of Forest ResE-mail address: hulcr@ufl.edu (J. Hulcr).

http://dx.doi.org/10.1016/j.funbio.2017.08.0041878-6146/ª 2017 British Mycological Society

a b s t r a c t

The ambrosia fungus Flavodon ambrosius is the primary nutritional mutualist of ambrosia

beetles Ambrosiodmus and Ambrosiophilus in North America. F. ambrosius is the only known

ambrosial basidiomycete, unique in its efficient lignocellulose degradation. F. ambrosius is

associated with both native American beetle species and species introduced from Asia. It re-

mains unknown whether F. ambrosius is strictly a North American fungus, or whether it is

also associated with these ambrosia beetle genera on other continents. We isolated fungi

from the mycangia and galleries of ambrosia beetles Ambrosiodmus rubricollis, Ambrosiodmus

minor, Ambrosiophilus atratus, and Ambrosiophilus subnepotulus in China, South Korea, and

Vietnam. Phylogenetic analyses suggest that all Asian and North American isolates repre-

sent a single haplotype. These results confirm Flavodon ambrosius as the exclusive mutualis-

tic fungus of multiple Ambrosiodmus and Ambrosiophilus beetle species around the world,

making it the most widespread known ambrosia fungus species, both geographically and

in terms of the number of beetle species. The Flavodon-beetle symbiosis appears to employ

an unusually strict mechanism for maintaining fidelity, compared to the symbioses of the

related Xyleborini beetles, which mostly vector more dynamic fungal communities.

ª 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction the beetles inoculate the newly-excavated tunnel systems

Ambrosia fungi are obligate nutritional mutualists of wood-

boring ambrosia beetles [Coleoptera: Curculionidae: Scolyti-

nae and Platypodinae (Batra 1963; Hulcr & Stelinski 2017)].

The fungi are carried by the beetles into new tree hosts, where

ources and Conservation

. Published by Elsevier L

with these fungi (Batra 1963 1966; Six 2003). Of at least ten in-

dependent origins of the symbiosis within Fungi, all known

ambrosia fungi lie within lineages of the phylum Ascomycota

(Alamouti et al. 2009; Kola�r�ık & Kirkendall 2010; Kasson et al.

2013; Dreaden et al. 2014; Mayers et al. 2015; Bateman et al.

, University of Florida, Gainesville, FL, USA. Tel.: þ1 352 2730299.

td. All rights reserved.

mailto:hulcr@ufl.eduhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.funbio.2017.08.004&domain=pdfhttp://www.elsevier.com/locate/funbiohttp://dx.doi.org/10.1016/j.funbio.2017.08.004http://dx.doi.org/10.1016/j.funbio.2017.08.004http://dx.doi.org/10.1016/j.funbio.2017.08.004

Wood decay fungus Flavodon ambrosius 985

unpublished data). The only known exception is the Flavodon

ambrosius D.R. Simmons, You Li, C.C. Bateman & Hulcr (Basi-

diomycota: Polyporales), which is not only the sole known ba-

sidiomycetous ambrosia fungus, but is also unique among

other ambrosia fungi in its capacity to efficiently utilize cellu-

lose and lignin (Kasson et al. 2016).

F. ambrosius is the primary mutualistic fungus of beetles in

the generaAmbrosiodmus andAmbrosiophilus (Scolytinae: Xyle-

borini) (Li et al. 2015; Kasson et al. 2016; Simmons et al. 2016).

These two beetle genera form a monophyletic clade (Storer

et al. 2014), suggesting a single origin of Flavodon farming.

Four beetle species have been recorded in association with F.

ambrosius: Ambrosiodmus lecontei, Ambrosiodmus minor, Ambro-

siodmus rubricollis (Li et al. 2015), and Ambrosiophilus atratus

(Kasson et al. 2016). All of these species, except A. lecontei,

were recently introduced to North America from Asia (Bright

1968; Atkinson et al. 1990; Wood & Bright 1992) and are now

established and common.

Many more species of Ambrosiodmus and Ambrosiophilus oc-

cur in Asia, Africa and South America, but their fungal symbi-

onts have either not been studied (Wood 2007; Beaver & Liu

2010; Kn�ı�zek 2011) or have been poorly identified (Yamashita

1966; Takagi 1967). Aside from F. ambrosius, the only described

species of Flavodon are the saprophytic and free-living polypore

species F. flavus (Klotzsch) Ryvarden (Ryvarden 1973), found in

southern Asia, tropical Africa to South Africa, Australia, and

Jamaica (Maas 1967; Ryvarden & Johansen 1980; Corner 1987;

Miettinen et al. 2012), and ‘F. cervinogilvus’ (Corner 1987), an in-

valid species first recorded from the eastern coast of the Island

of Hawaii (Simmons et al. 2016). It is therefore unclear whether

Asian Ambrosiodmus and Ambrosiophilus 1) also farm F. ambro-

sius, 2) farm a different symbiotic species of Flavodon, or 3)

farm a non-wood-decaying Ascomycete fungus species, as

would be far more typical among ambrosia beetles.

The goal of this work was to test whether Asian Ambrosio-

philus and Ambrosiodmus ambrosia beetle are associated with

F. ambrosius in their native regions. The possibility of F. ambro-

sius playing the same symbiotic role around the worldmay in-

dicate a scenario of unusual dominance of a single ambrosia

fungus over many beetle species separated by continental-

scale barriers and about 20 Ma (Jordal & Cognato 2012). Such

strict associations between the Ambrosiodmus/Ambrosiophilus

clade and their fungi would be in contrast to the related line-

ages of ambrosia beetles, namely Xyleborus and Euwallacea,

most of which carry diverse and often loose symbiotic fungi

in both native and non-native regions (Harrington et al. 2010;

Hulcr & Cognato 2010; Kostovcik et al. 2015; O’Donnell et al.

2015; Ploetz et al. 2017). The results expand our understanding

of the symbiotic relationship between basidiomycetouswood-

decaying ambrosia fungi and their beetle vectors.

Materials and methods

Two Ambrosiodmus and two Ambrosiophilus species from Asia

were sampled in four locations throughout China, South

Korea, and Vietnam (October 2015 through May 2016;

Table 1). All beetles were excised directly from active galleries

in wood. In addition to gallery excision, Ambrosiophilus atratus

was also caught in ethanol-baited traps in South Korea. To

ensure finding the actual primary symbiont of each beetle

species, we focused on fungi isolated strictly from the oral

mycangia (excised by cutting off the frontal part of the head)

and from active galleries of Ambrosiodmus and Ambrosiophilus

beetles. Cultures obtained from beetle hosts were isolated by

dilution-plating of mycangial contents and gallery wood chips

on PDA medium, as described by Li et al. (2015). Colony form-

ing units (CFU) of fungal isolation were recorded. Pure isolates

were imported into, and studied in, a quarantine facility in

Gainesville, FL, USA, under the USDA/APHIS permit No.

P526P-16-02872. Beetle and fungus voucher specimens are

preserved and stored at the Forest Entomology Laboratory,

School of Forest Resources and Conservation, University of

Florida, Gainesville, FL, USA.

DNA was extracted from pure subcultures. Sequences of

the nuclear internal transcribed spacers ITS1-5.8S-ITS2 (ITS)

and nuclear 28S ribosomal DNA (rDNA) regions were ampli-

fied. The primer pairs for PCR amplification were ITS1/ITS4

(White et al. 1990) and LROR/LR5 (Vilgalys & Hester 1990).

PCR amplification of ITS and 28S followed protocols of Li

et al. (2015). Sequencing of the PCR products in forward and re-

verse directions, and editing and assembling of the nucleotide

sequenceswere accomplished following Simmons et al. (2016).

To place Asian Flavodon isolateswithin the knownphyloge-

netic context, we selected 28S rDNA sequences fromSimmons

et al. (2016) and ITS rDNA sequences from Li et al. (2015),

Kasson et al. (2016) and Miettinen et al. (2012), which included

Flavodon ambrosius and related taxa. We aligned these se-

quences with those from our cultured Ambrosiodmus and

Ambrosiophilus-associated fungi using default settings in Clus-

talX 2.0 (www.clustal.org; Hall 2013). The alignments are de-

posited in TreeBASE (Reviewer access URL: http://purl.org/

phylo/treebase/phylows/study/TB2:S20605?x-access-code-

¼a5b8cb957aff2c80c1985d844175f6ee&format¼html). Se-quence evolution model for the Maximum likelihood (ML)

analyses was selected using Mega 7 (Hall 2013) setting the ini-

tial tree for ML calculations to NJ/BioNJ. Tamura 3 þ G was se-lected for the ITS dataset and Kimura 2 þ G model wasselected for the 28S dataset. ML analyses were also conducted

with Mega 7, setting the number of fast bootstrap (BS) repli-

cates to 1000.

Results and discussion

The unique ambrosia fungus Flavodon ambrosius was so far

studied exclusively in the U.S. (Li et al. 2015; Kasson et al.

2016) but the majority of its vectors in the beetle genera

Ambrosiodmus and Ambrosiophilus are not native in North

America, and their greatest diversity occurs in Asia (Wood

2007; Kn�ı�zek 2011). Our goal was to study whether these bee-

tles also employ F. ambrosius as their primary symbiotic fun-

gus in Asia where they are indigenous.

Identification and phylogenetic analyses of symbiotic fungusFlavodon ambrosius

TheML phylogenetic analyses of both the ITS and 28Smarkers

recovered nearly identical topology for the Flavodon clade

(Fig. 1). The topology corroborated the placement of Flavodon

http://www.clustal.orghttp://purl.org/phylo/treebase/phylows/study/TB2:S20605?x-access-code=a5b8cb957aff2c80c1985d844175f6ee&format=htmlhttp://purl.org/phylo/treebase/phylows/study/TB2:S20605?x-access-code=a5b8cb957aff2c80c1985d844175f6ee&format=htmlhttp://purl.org/phylo/treebase/phylows/study/TB2:S20605?x-access-code=a5b8cb957aff2c80c1985d844175f6ee&format=htmlhttp://purl.org/phylo/treebase/phylows/study/TB2:S20605?x-access-code=a5b8cb957aff2c80c1985d844175f6ee&format=html

Table 1 e The primary symbiont isolated from Asian Ambrosiodmus and Ambrosiophilus beetles and the NCBI/GenBankaccession numbers of their ITS and 28S rDNA sequences. CFU: the average count of fungus cells in the mycangiumcalculated as the average count of colony-forming units on culture plates multiplied by the dilution factor.

Taxon Voucher no. Locality Beetle vector CFU Isolationbody part

GenBankaccession no.

ITS 28S

Flavodon ambrosius LL51 Huaxi, Guizhou, China Ambrosiodmus rubricollis 16000 oral mycangia LC216225 LC215903

F. ambrosius LL53 Huaxi, Guizhou, China Ambrosiodmus rubricollis N/A gallery LC216226 LC215904

F. ambrosius LL70 Nanming, Guizhou, China Ambrosiodmus rubricollis 500 oral mycangia LC216227 LC215905

F. ambrosius LL71 Nanming, Guizhou, China Ambrosiophilus subnepotulus 1500 oral mycangia LC216228 LC215906

F. ambrosius V12236 Tam Ðảo, Vietnam Ambrosiodmus minor 1500 oral mycangia LC216229 LC215907F. ambrosius V12544 Gwangyang, South Korea Ambrosiophilus atratus 1100 oral mycangia LC216230 LC215908

F. ambrosius V12546 Gwangyang, South Korea Ambrosiophilus atratus 1000 oral mycangia LC216231 LC215909

986 Y. Li et al.

ambrosius in the genus Flavodon together with F. flavus in the

phlebioid clade in the Polyporales (Li et al. 2015). All Asian Fla-

vodon sequences were placed within the homogeneous F.

ambrosius clade, which consisted of sequences from the F.

ambrosius holotype and other isolates from Florida and West

Virginia, from both native and non-native North American

beetle species. The F. ambrosius sequences did not show any

divergence or lineages specific to different beetle vectors or

locations.

The monophyly of all F. ambrosius isolates studied here

strongly supports the hypothesis that beetles in the Ambro-

siodmus/Ambrosiophilus clade share the same symbiotic fungus

in Asia and North America. In total, five ambrosia beetle spe-

cies in Ambrosiophilus and Ambrosiodmus are now confirmed to

employ F. ambrosius as their primary symbiotic fungus (Li et al.

2015; Kasson et al. 2016; this study). The symbiosis between F.

ambrosius and all its vectors has beenmaintained in both deep

evolutionary time during the natural spread of ancestral

Ambrosiodmus around the world, as well as during recent

human-assisted spread and introductions.

The monophyly of F. ambrosius in two markers commonly

used in fungal systematics indicates that all the isolates are

members of the same phylogenetic species. The ITS region is

typically variable enough to detect sub-species divergence in

fungi of Basidiomycota (Lindner & Banik 2011; Schoch et al.

2012), but it did not recover any divergence within F.

Fig 1 e The best Maximum likelihood tree of Flavodon inferred fro

GenBank accession numbers. Values above nodes represent ML

replicates. The beetle vector genera are indicated by squares an

ambrosius. If multiple lineages exist within F. ambrosius, they

are either unrecoverable using the two markers selected

here, or additional Flavodon diversity occurs in geographic re-

gions not sampled for this work, such as in South America.

All Flavodon strains isolated in this work were morpholog-

ically uniform. F. ambrosius isolated from Ambrosiophilus atra-

tus in West Virginia displayed two types of colony

morphology, but both were genetically identical (Kasson

et al. 2016). An ambrosia fungus that was likely Flavodon was

previously recorded from Ambrosiodmus rubricollis from Japan

(Yamashita 1966; Tagaki 1967) and described as ‘white wooly

mycelia’ without further identification. Isolates of this fungus

were not available to us, but the brief description fits the mor-

phology of F. ambrosius.

Most ambrosia beetles maintain their symbiont associa-

tions through time and through introductions into non-

native regions. Mayers et al. (2015) suggests that in some

groups, each beetle species is associated with a unique fungus

species, and each fungus is typically only found associated

with one beetle species. In other beetle clades, co-

phylogenetic analysis has suggested that symbiotic partners

may be frequently swapped over evolutionary time

(O’Donnell et al. 2015). The association between Ambrosiod-

mus/Ambrosiophilus and F. ambrosius appears to represent an

entirely different scenario. Our results suggest an association

of a single fungus and many beetle species, a dominance of

m ITS and 28S rDNA datasets with source beetle genera and

bootstrap percentages >50 % from a summary of 1000

d circles.

Wood decay fungus Flavodon ambrosius 987

a single fungus that has been maintained throughout evolu-

tionary history as the beetles have radiated into many species

and spread around the world. Why has F. ambrosius not also

radiated into diverse lineages of fungal symbionts that are

each specialized for its own vector beetle, as has been ob-

served in other closely related ambrosia beetle clades? Could

the unique ecological traits of this sole known white-rotting

basidiomycete ambrosia fungus explain these unique rela-

tionships? These questions offer avenues for future enquiry

Fig 2 e Flavodon ambrosius and ambrosia beetles collected from C

(B) Gallery of Ambrosiophilus subnepotuluswith F. ambrosius. (C) B

rubricollis (right and left) on the bark. (D) Galleries of Ambrosioph

the same branch; Red arrows: F. ambrosius; Bars: 1 mm. (For int

the reader is referred to the web version of this article.)

and remain at the forefront of symbiosis and co-evolutio-

nary research.

Biology of fungus Flavodon ambrosius

The incidence and habits of F. ambrosius in Asia were consis-

tent with those observed in North America. All branches con-

taining F. ambrosius and their beetle vectors were notably

decayed (Fig 2A). In many cases, both Ambrosiodmus and

hina. (A) White-rot wood with galleries of ambrosia beetles.

oring holes of Ambrosiophilus sp. (middle) and Ambrosiodmus

ilus subnepotulus (right) and Ambrosiodmus rubricollis (left) on

erpretation of the references to colour in this figure legend,

988 Y. Li et al.

Ambrosiophilus tunnel entrances were closely clustered,

rather than being randomly distributed on the tree. For ex-

ample, the F. ambrosius samples from China were collected

frommultiple galleries of Ambrosiodmus rubricollis andAmbro-

siophilus subnepotulus found immediately adjacent to one an-

other on the same branch. Galleries of both beetle species

were covered by white filamentous fungal growth (Fig 2D).

These observations support the hypothesis that some Ambro-

siophilus species are mycocleptic, colonizing the vicinity of

galleries established by other ambrosia beetles carrying the

same fungus (Fig 2B and C; Hulcr & Cognato 2010). The one

species of Ambrosiophilus that has been well-studied (Ambro-

siophilus atratus) has oral mycangia with the capacity to trans-

mit and preserve fungal inoculum, and frequently creates its

own galleries (Kasson et al. 2016). However, the Ambrosiophi-

lus species which are more often reported as mycocleptic

have not been studied for their mycangia, and are typically

found associated with ambrosia beetles from the genus Bea-

verium (Hulcr & Cognato 2010). This is the first report of mul-

tiple mycocleptic association between Ambrosiophilus and

Ambrosiodmus.

The evolutionary transition of Flavodon from a free-living

species to the ambrosial phenotype seen in F. ambrosius de-

serves future study. As in the USA, the Asian F. ambrosius is

also known only from ambrosia beetles, and no fruiting

body has been reported (Li et al. 2015; Kasson et al. 2016;

Simmons et al. 2016). The closely related free-living species

F. flavus is common in tropical regions of Asia, and frequently

produces sexual fruiting bodies (Dai 2012). Our data do not al-

low to ascertainwhether F. ambrosius participates in sexual re-

production, or whether the fungus is horizontally acquired by

the beetles from free-living populations, but neither has been

reported.

Conclusions

This study suggests that a single species of the white-rot ba-

sidiomycete ambrosia fungus Flavodon ambrosius is the pri-

mary associate of all Ambrosiodmus and Ambrosiophilus

ambrosia beetles in the northern hemisphere. Many unex-

plored species of Ambrosiodmus occur throughout Africa and

South America, but they were not available during this pro-

ject. In addition, the genus Beaverium, widespread in Asia

and Oceania, is also likely associated with Flavodon, as sug-

gested by the frequent co-colonization by Ambrosiophilus. If

all these beetles indeed carry F. ambrosius as their symbiont,

it would make this fungus one of the most widespread fungal

symbionts of animals.

Acknowledgements

We gratefully acknowledge Jian-jun Guo (Guizhou University,

China), Ki-Jeong Hong andMoo-Sung Kim (Suncheon National

University), Pham Hong Thai and Tran Thi Men (Vietnam Na-

tional Museum of Nature) for their assistance in sample col-

lecting and logistic support. Dr. Roger Beaver and Lan-yu Liu

(National Museum of Natural Sciences, Taiwan) helped us

identify Ambrosiophilus subnepotulus. The study was supported

by the USDA Forest Service, USDA Farm Bill section 100007, the

Florida Department of Agriculture and Consumer Services e

Division of Plant Industry, and the National Science

Foundation.

r e f e r e n c e s

Alamouti SM, Tsui CK, Breuil C, 2009. Multigene phylogeny offilamentous ambrosia fungi associated with ambrosia andbark beetles. Mycological Research 113: 822e835.

Atkinson TH, Rabaglia RJ, Bright DE, 1990. Newly detected exoticspecies of Xyleborus (Coleoptera: Scolytidae) with a revised keyto species in eastern North America. The Canadian Entomologist122: 93e104. http://dx.doi.org/10.4039/Ent12293-1.

Batra LR, 1963. Ecology of ambrosia fungi and their disseminationby beetles. Transactions of the Kansas Academy of Science 66:213e236.

Batra LR, 1966. Ambrosia fungi: extent of specificity to ambrosiabeetles. Science 153: 193e195.

Beaver R, Liu L, 2010. An annotated synopsis of Taiwanese barkand ambrosia beetles, with new synonymy, new combina-tions and new records (Coleoptera: Curculionidae: Scolytinae).Zootaxa 2602: 1e47.

Bright DEJ, 1968. Review of the Tribe Xyleborini in America northof Mexico (Coleoptera: Scolytidae). Canadian Entomologist 100:1288e1323.

Corner E, 1987. Ad Polyporaceae IV. Beihefte Zur Nova Hedwigia 86:1e265.

Dai YC, 2012. Polypore diversity in China with an annotatedchecklist of Chinese polypores. Mycoscience 53: 49e80. http://dx.doi.org/10.1007/s10267-011-0134-3.

Dreaden TJ, Davis JM, de Beer ZW, Ploetz RC, Soltis PS,Wingfield MJ, Smith JA, 2014. Phylogeny of ambrosia beetlesymbionts in the genus Raffaelea. Fungal Biology 118: 970e978.

Hall BG, 2013. Building phylogenetic trees from molecular datawith MEGA. Molecular Biology and Evolution 30: 1229e1235.

Harrington T, Aghayeva D, Fraedrich S, 2010. New combinationsin Raffaelea, Ambrosiella, and Hyalorhinocladiella, and four newspecies from the redbay ambrosia beetle, Xyleborus glabratus.Mycotaxon 111: 337e361.

Hulcr J, Cognato AI, 2010. Repeated evolution of crop theft infungus-farming ambrosia beetles. Evolution 64: 3205e3212.http://dx.doi.org/10.1111/j.1558-5646.2010.01055.x.

Hulcr J, Stelinski LL, 2017. The ambrosia symbiosis: from evolu-tionary ecology to practical management. Annual Review ofEntomology 62: 285e303. http://dx.doi.org/10.1146/annurev-ento-031616-035105.

Jordal BH, Cognato AI, 2012. Molecular phylogeny of bark andambrosia beetles reveals multiple origins of fungus farmingduring periods of global warming. BMC Evolutionary Biology 12:133. http://dx.doi.org/10.1186/1471-2148-12-133.

Kasson MT, O’Donnell K, Rooney AP, Sink S, Ploetz RC, Ploetz JN,Konkol JL, Carrillo D, Freeman S, Mendel Z, Smith JA,Black AW, Hulcr J, Bateman C, Stefkova K, Campbell PR,Geering ADW, Dann EK, Eskalen A, Mohotti K, Short DPG,Aoki T, Fenstermacher KA, Davis DD, Geiser DM, 2013. An in-ordinate fondness for Fusarium: phylogenetic diversity of fu-saria cultivated by ambrosia beetles in the genus Euwallaceaon avocado and other plant hosts. Fungal Genetics and Biology56: 147e157. http://dx.doi.org/10.1016/j.fgb.2013.04.004.

Kasson MT, Wickert KL, Stauder CM, Macias AM, Berger MC,Simmons DR, Short DP, DeVallance DB, Hulcr J, 2016. Mutu-alism with aggressive wood-degrading Flavodon ambrosius(Polyporales) facilitates niche expansion and communal socialstructure in Ambrosiophilus ambrosia beetles. Fungal Ecology23: 86e96. http://dx.doi.org/10.1016/j.funeco.2016.07.002.

http://refhub.elsevier.com/S1878-6146(17)30107-1/sref2http://refhub.elsevier.com/S1878-6146(17)30107-1/sref2http://refhub.elsevier.com/S1878-6146(17)30107-1/sref2http://refhub.elsevier.com/S1878-6146(17)30107-1/sref2http://dx.doi.org/10.4039/Ent12293-1http://refhub.elsevier.com/S1878-6146(17)30107-1/sref4http://refhub.elsevier.com/S1878-6146(17)30107-1/sref4http://refhub.elsevier.com/S1878-6146(17)30107-1/sref4http://refhub.elsevier.com/S1878-6146(17)30107-1/sref4http://refhub.elsevier.com/S1878-6146(17)30107-1/sref5http://refhub.elsevier.com/S1878-6146(17)30107-1/sref5http://refhub.elsevier.com/S1878-6146(17)30107-1/sref5http://refhub.elsevier.com/S1878-6146(17)30107-1/sref6http://refhub.elsevier.com/S1878-6146(17)30107-1/sref6http://refhub.elsevier.com/S1878-6146(17)30107-1/sref6http://refhub.elsevier.com/S1878-6146(17)30107-1/sref6http://refhub.elsevier.com/S1878-6146(17)30107-1/sref6http://refhub.elsevier.com/S1878-6146(17)30107-1/sref7http://refhub.elsevier.com/S1878-6146(17)30107-1/sref7http://refhub.elsevier.com/S1878-6146(17)30107-1/sref7http://refhub.elsevier.com/S1878-6146(17)30107-1/sref7http://refhub.elsevier.com/S1878-6146(17)30107-1/sref8http://refhub.elsevier.com/S1878-6146(17)30107-1/sref8http://refhub.elsevier.com/S1878-6146(17)30107-1/sref8http://dx.doi.org/10.1007/s10267-011-0134-3http://dx.doi.org/10.1007/s10267-011-0134-3http://refhub.elsevier.com/S1878-6146(17)30107-1/sref10http://refhub.elsevier.com/S1878-6146(17)30107-1/sref10http://refhub.elsevier.com/S1878-6146(17)30107-1/sref10http://refhub.elsevier.com/S1878-6146(17)30107-1/sref10http://refhub.elsevier.com/S1878-6146(17)30107-1/sref12http://refhub.elsevier.com/S1878-6146(17)30107-1/sref12http://refhub.elsevier.com/S1878-6146(17)30107-1/sref12http://refhub.elsevier.com/S1878-6146(17)30107-1/sref14http://refhub.elsevier.com/S1878-6146(17)30107-1/sref14http://refhub.elsevier.com/S1878-6146(17)30107-1/sref14http://refhub.elsevier.com/S1878-6146(17)30107-1/sref14http://refhub.elsevier.com/S1878-6146(17)30107-1/sref14http://dx.doi.org/10.1111/j.1558-5646.2010.01055.xhttp://dx.doi.org/10.1146/annurev-ento-031616-035105http://dx.doi.org/10.1146/annurev-ento-031616-035105http://dx.doi.org/10.1186/1471-2148-12-133http://dx.doi.org/10.1016/j.fgb.2013.04.004http://dx.doi.org/10.1016/j.funeco.2016.07.002

Wood decay fungus Flavodon ambrosius 989

Kn�ı�zek M, 2011. Subfamily Scolytinae Latreille, 1804. In: L€obl I,Smetana A (eds), Catalogue of Palaearctic Coleoptera. Part I.Stenstrup, vol. 7. Apollo Books, Denmark, p. 373.

Kola�r�ık M, Kirkendall LR, 2010. Evidence for a new lineage ofprimary ambrosia fungi in Geosmithia Pitt (Ascomycota: Hy-pocreales). Fungal Biology 114: 676e689.

Kostovcik M, Bateman CC, Kolarik M, Stelinski LL, Jordal BH,Hulcr J, 2015. The ambrosia symbiosis is specific in somespecies and promiscuous in others: evidence from communitypyrosequencing. The ISME Journal 9: 126e138.

Li Y, Simmons RD, Bateman CC, Short DP, Kasson MT, Rabaglia RJ,Hulcr J, 2015. New fungus-insect symbiosis: culturing, molec-ular, and histological methods determine saprophytic Poly-porales mutualists of Ambrosiodmus ambrosia beetles. PloS One10: e0137689. http://dx.doi.org/10.1371/journal.pone.0137689.

Lindner DL, Banik MT, 2011. Intragenomic variation in the ITSrDNA region obscures phylogenetic relationships and inflatesestimates of operational taxonomic units in genus Laetiporus.Mycologia 103: 731e740.

Maas Geesteranus RA, 1967. Quelques champignons hydno€ıdesdu Congo. Bulletin du Jardin botanique national de Belgique/Bulle-tin van de Nationale Plantentuin van Belgie 77e107.

Mayers CG, McNew DL, Harrington TC, Roeper RA, Fraedrich SW,Biedermann PH, Castrillo LA, Reed SE, 2015. Three genera inthe Ceratocystidaceae are the respective symbionts of threeindependent lineages of ambrosia beetles with large, complexmycangia. Fungal Biology 119: 1075e1092.

Miettinen O, Larsson E, Sj€okvist E, Larsson KH, 2012. Compre-hensive taxon sampling reveals unaccounted diversity andmorphological plasticity in a group of dimitic polypores (Pol-yporales, Basidiomycota). Cladistics 28: 251e270.

O’Donnell K, Libeskind-Hadas R, Hulcr J, Bateman C, Kasson MT,Ploetz RC, Carrillo D, Campbell A, Duncan RE, Liyanage PNH,Eskalen A, Lynch SC, Geiser DM, Freeman S, Mendel Z,Sharon M, Aoki T, Coss�e AA, Rooney AP, 2015. Invasive AsianFusarium e Euwallacea ambrosia beetle mutualists pose a seri-ous threat to forests, urban landscapes and the avocado in-dustry. Phytoparasitica 44: 435e442.

Ploetz RC, Konkol JL, Narvaez T, Duncan RE, Saucedo RJ,Campbell A, Mantilla J, Carrillo D, Kendra PE, 2017. Presenceand prevalence of Raffaelea lauricola, cause of laurel wilt, in

different species of ambrosia beetle in Florida, USA. Journal ofEconomic Entomology 110: 1e8.

Ryvarden L, 1973. New genera in the Polyporaceae. NorwegianJournal of Botany 20: 1e5.

Ryvarden L, Johansen I, 1980. A Preliminary Polypore Flora of EastAfrica. Fungiflora, Oslo.

Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL,Levesque CA, Chen W, Bolchacova E, Voigt K, Crous PW,Miller AN, 2012. Nuclear ribosomal internal transcribed spacer(ITS) region as a universal DNA barcode marker for Fungi.PNAS 109: 6241e6246.

Simmons DR, Li Y, Bateman CC, Hulcr J, 2016. Flavodon ambrosiussp. nov., a basidiomycetous mycosymbiont of Ambrosiodmusambrosia beetles. Mycotaxon 131: 277e285.

Six DL, 2003. Bark beetle-fungus symbioses. In: Bourtzis K,Miller TA (eds), Insect Symbiosis, vol. 1. CRC Press, Washington,D.C, pp. 97e114.

Storer CG, Breinholt JW, Hulcr J, 2014. Wallacellus is Euwallacea:molecular phylogenetics settles generic relationships (Cole-optera: Curculionidae: Scolytinae: Xyleborini). Zootaxa 3974:391e400.

Takagi K, 1967. The storage organ of symbiotic fungus in theambrosia beetle Xyleborus rubricollis EICHHOFF (Coleoptera:Scolytidae). Japanese Society of Applied Entomology and Zoology 2:168e170.

Vilgalys R, Hester M, 1990. Rapid genetic identification and map-ping of enzymatically amplified ribosomal DNA from severalCryptococcus species. Journal of Bacteriology 172: 4238e4246.

White TJ, Bruns T, Lee S, Taylor J, 1990. Amplification and directsequencing of fungal ribosomal RNA genes for phylogenetics.In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Pro-tocols: a guide to methods and applications. Academic Press, USA,pp. 315e322.

Wood SL, 2007. Bark and ambrosia beetles of South America(Coleoptera, Scolytidae). Brigham Young University Press.

Wood SL, Bright DEJ, 1992. A catalog of Scolytidae and Platypodidae(Coleoptera), Part 2. Taxonomic Index. Great Basin NaturalistMemoirs, Utah.

Yamashita M, 1966. Preliminary report on artificial rearing ofXyleborus rubricollis EICHHOFF attacking Chestnut-trees. Japa-nese Journal of Applied Entomology and Zoology 10: 95e96.

http://refhub.elsevier.com/S1878-6146(17)30107-1/sref21http://refhub.elsevier.com/S1878-6146(17)30107-1/sref21http://refhub.elsevier.com/S1878-6146(17)30107-1/sref21http://refhub.elsevier.com/S1878-6146(17)30107-1/sref21http://refhub.elsevier.com/S1878-6146(17)30107-1/sref21http://refhub.elsevier.com/S1878-6146(17)30107-1/sref22http://refhub.elsevier.com/S1878-6146(17)30107-1/sref22http://refhub.elsevier.com/S1878-6146(17)30107-1/sref22http://refhub.elsevier.com/S1878-6146(17)30107-1/sref22http://refhub.elsevier.com/S1878-6146(17)30107-1/sref22http://refhub.elsevier.com/S1878-6146(17)30107-1/sref23http://refhub.elsevier.com/S1878-6146(17)30107-1/sref23http://refhub.elsevier.com/S1878-6146(17)30107-1/sref23http://refhub.elsevier.com/S1878-6146(17)30107-1/sref23http://refhub.elsevier.com/S1878-6146(17)30107-1/sref23http://dx.doi.org/10.1371/journal.pone.0137689http://refhub.elsevier.com/S1878-6146(17)30107-1/sref25http://refhub.elsevier.com/S1878-6146(17)30107-1/sref25http://refhub.elsevier.com/S1878-6146(17)30107-1/sref25http://refhub.elsevier.com/S1878-6146(17)30107-1/sref25http://refhub.elsevier.com/S1878-6146(17)30107-1/sref25http://refhub.elsevier.com/S1878-6146(17)30107-1/sref26http://refhub.elsevier.com/S1878-6146(17)30107-1/sref26http://refhub.elsevier.com/S1878-6146(17)30107-1/sref26http://refhub.elsevier.com/S1878-6146(17)30107-1/sref26http://refhub.elsevier.com/S1878-6146(17)30107-1/sref26http://refhub.elsevier.com/S1878-6146(17)30107-1/sref27http://refhub.elsevier.com/S1878-6146(17)30107-1/sref27http://refhub.elsevier.com/S1878-6146(17)30107-1/sref27http://refhub.elsevier.com/S1878-6146(17)30107-1/sref27http://refhub.elsevier.com/S1878-6146(17)30107-1/sref27http://refhub.elsevier.com/S1878-6146(17)30107-1/sref27http://refhub.elsevier.com/S1878-6146(17)30107-1/sref29http://refhub.elsevier.com/S1878-6146(17)30107-1/sref29http://refhub.elsevier.com/S1878-6146(17)30107-1/sref29http://refhub.elsevier.com/S1878-6146(17)30107-1/sref29http://refhub.elsevier.com/S1878-6146(17)30107-1/sref29http://refhub.elsevier.com/S1878-6146(17)30107-1/sref29http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref30http://refhub.elsevier.com/S1878-6146(17)30107-1/sref31http://refhub.elsevier.com/S1878-6146(17)30107-1/sref31http://refhub.elsevier.com/S1878-6146(17)30107-1/sref31http://refhub.elsevier.com/S1878-6146(17)30107-1/sref31http://refhub.elsevier.com/S1878-6146(17)30107-1/sref31http://refhub.elsevier.com/S1878-6146(17)30107-1/sref31http://refhub.elsevier.com/S1878-6146(17)30107-1/sref32http://refhub.elsevier.com/S1878-6146(17)30107-1/sref32http://refhub.elsevier.com/S1878-6146(17)30107-1/sref32http://refhub.elsevier.com/S1878-6146(17)30107-1/sref33http://refhub.elsevier.com/S1878-6146(17)30107-1/sref33http://refhub.elsevier.com/S1878-6146(17)30107-1/sref34http://refhub.elsevier.com/S1878-6146(17)30107-1/sref34http://refhub.elsevier.com/S1878-6146(17)30107-1/sref34http://refhub.elsevier.com/S1878-6146(17)30107-1/sref34http://refhub.elsevier.com/S1878-6146(17)30107-1/sref34http://refhub.elsevier.com/S1878-6146(17)30107-1/sref34http://refhub.elsevier.com/S1878-6146(17)30107-1/sref35http://refhub.elsevier.com/S1878-6146(17)30107-1/sref35http://refhub.elsevier.com/S1878-6146(17)30107-1/sref35http://refhub.elsevier.com/S1878-6146(17)30107-1/sref35http://refhub.elsevier.com/S1878-6146(17)30107-1/sref36http://refhub.elsevier.com/S1878-6146(17)30107-1/sref36http://refhub.elsevier.com/S1878-6146(17)30107-1/sref36http://refhub.elsevier.com/S1878-6146(17)30107-1/sref36http://refhub.elsevier.com/S1878-6146(17)30107-1/sref37http://refhub.elsevier.com/S1878-6146(17)30107-1/sref37http://refhub.elsevier.com/S1878-6146(17)30107-1/sref37http://refhub.elsevier.com/S1878-6146(17)30107-1/sref37http://refhub.elsevier.com/S1878-6146(17)30107-1/sref37http://refhub.elsevier.com/S1878-6146(17)30107-1/sref38http://refhub.elsevier.com/S1878-6146(17)30107-1/sref38http://refhub.elsevier.com/S1878-6146(17)30107-1/sref38http://refhub.elsevier.com/S1878-6146(17)30107-1/sref38http://refhub.elsevier.com/S1878-6146(17)30107-1/sref38http://refhub.elsevier.com/S1878-6146(17)30107-1/sref40http://refhub.elsevier.com/S1878-6146(17)30107-1/sref40http://refhub.elsevier.com/S1878-6146(17)30107-1/sref40http://refhub.elsevier.com/S1878-6146(17)30107-1/sref40http://refhub.elsevier.com/S1878-6146(17)30107-1/sref42http://refhub.elsevier.com/S1878-6146(17)30107-1/sref42http://refhub.elsevier.com/S1878-6146(17)30107-1/sref42http://refhub.elsevier.com/S1878-6146(17)30107-1/sref42http://refhub.elsevier.com/S1878-6146(17)30107-1/sref42http://refhub.elsevier.com/S1878-6146(17)30107-1/sref42http://refhub.elsevier.com/S1878-6146(17)30107-1/sref45http://refhub.elsevier.com/S1878-6146(17)30107-1/sref45http://refhub.elsevier.com/S1878-6146(17)30107-1/sref43http://refhub.elsevier.com/S1878-6146(17)30107-1/sref43http://refhub.elsevier.com/S1878-6146(17)30107-1/sref43http://refhub.elsevier.com/S1878-6146(17)30107-1/sref44http://refhub.elsevier.com/S1878-6146(17)30107-1/sref44http://refhub.elsevier.com/S1878-6146(17)30107-1/sref44http://refhub.elsevier.com/S1878-6146(17)30107-1/sref44

Wood decay fungus Flavodon ambrosius (Basidiomycota: Polyporales) is widely farmed by two genera of ambrosia beetlesIntroductionMaterials and methodsResults and discussionIdentification and phylogenetic analyses of symbiotic fungus Flavodon ambrosiusBiology of fungus Flavodon ambrosius

ConclusionsAcknowledgementsReferences